Cell separation apparatus and methods of use

ABSTRACT

Cell separation systems and methods of separating cells are disclosed. In an embodiment, a cell separation system is described that comprises a non-transitory storage device that executes a centrifugation program to separate cell volume from biologic material volume; a heating mechanism; a containment mechanism; and an assembly comprised of a single-walled centrifugation bowl. In an embodiment, methods of separating cells are disclosed whereby cells are separated by agitating a volume of biologic material and a volume digestion media to form a digested volume of biologic material; centrifuging the digested volume of biologic material; removing a portion of a resulting waste via at least one fluid outlet; isolating a different portion of the waste, and removing the concentrated cell volumes from the reservoir.

RELATED APPLICATION INFORMATION

This application claims priority and is a continuation-in-part of U.S.application Ser. No. 14/868,266, filed on Sep. 28, 2015 and which is adivisional of U.S. application Ser. No. 11/789,188, filed on Apr. 23,2007, titled CELL SEPARATION APPARATUS AND METHODS OF USE, and thatissued as U.S. Pat. No. 9,144,583, each of which are incorporated hereinby reference in its entirety.

BACKGROUND

Cell therapy and tissue engineering is developing toward clinicalapplications for the repair and restoration of damaged or diseasedtissues and organs. In particular, the development of tissue graftspromotes developments in surgeries, including cardiac and peripheralvascular surgery, limb tissue repair, dental applications, as well asveterinary surgeries. Grafts and other cell-based products may be formedby isolating and/or culturing cells from human or animal tissue.

Researchers have been studying synthetic grafts for over 30 years. Amajor challenge is providing graft materials that are biocompatible.i.e., non-thrombogenic, non-immunogenic, mechanically resistant, andhave acceptable wound healing and physiological responses (e.g.,vasoconstriction/relaxation responses, solute transportation ability,etc.). Furthermore, tissue graft materials should be easy to handle,store and ship, and be commercially feasible.

Vessels have two principal failure modes: mechanical and biological,caused by thrombosis within the vessel and subsequent occlusion and/orcellular ingrowth. Synthetic vessels having material properties capableof withstanding arterial pressure are commonplace, making the search fornon-thrombogenic materials the prime research interest. Endothelialcells obtained from the patient have been shown to decrease thethrombogenicity of implanted vessels.

Pressure gradients involving transient high pressures have been used todeposit cells onto a permeable scaffold by a sieving action, i.e.,providing a bulk flow and using a substrate or scaffold material havingpores smaller than the cell population, thus capturing cells in thematrix. These captured cells have been shown to subsequently adhere tothe scaffold material, but with only limited clinical applicability dueto failure to fully meet the requisites for successful grafts discussedabove, i.e., biocompatibility, mechanical strength, and necessaryphysiological properties.

Beginning in the late 1970s, endothelial cell seeding was employedexperimentally to improve the patency of small diameter, polymericvascular grafts to counteract adverse reactions. Since that time,advances have been made toward this goal, with the majority of the focuson engineering a biological or a bio-hybrid graft.

Endothelial cells are complex in that they do not merely create a singlecell lining on the lumenal surface of blood vessels. Endothelial cellsalso release molecules that modulate coagulation, platelet aggregation,leukocyte adhesion, and vascular tone. In the absence of these cells,e.g., in the case of the lumen of an implanted synthetic polymericvascular graft, the host reaction progresses to eventual failure. Lossof patency within the first thirty days post-implantation is due toacute thrombosis. This early stage failure is a consequence of theinherent thrombogenicity of the biomaterial's blood-contacting surface,which is non-endothelialized. To date, the only known completelynon-thrombogenic material is an endothelium; any other material thatcomes into contact with the bloodstream is predisposed to plateletdeposition and subsequent thrombosis. The long-term failure mode ofsmall diameter polymeric vascular grafts is anastomotic hyperplasialeading to a loss of patency. The precise mechanisms behind initiationof anastomotic hyperplasia are still being defined; however, endothelialcell and smooth muscle cell dysfunctions and improper communications arelikely involved.

Early workers in the field of small diameter graft development sought topromote graft endothelialization and, thereby, increase patency bytransplanting a varying degree of autologous endothelial cells ontovascular grafts prior to implantation. This process has become known asendothelial cell seeding (partial coverage relying on continued cellproliferation) or cell sodding (full coverage). “Seeding” refers to aprocess which includes preclotting prosthetic surfaces with endothelialcells in platelet rich plasma (PRP). Sodding, by comparison, refers to aprocess which includes plating endothelial cells onto a pre-establishedPRP clot. Sodded graft surfaces are typically prepared utilizing atwo-step procedure. First, PRP is clotted onto a graft, incubated for aneffective period of time and then washed with culture media. Second, thePRP coated graft is plated with endothelial cells. In contrast, seededgraft surfaces are typically prepared using a one-step platingprocedure, whereby endothelial cells suspended directly in PRP areplated onto a graft surface. Accordingly, in a sodded graft, endothelialcells are plated onto the surface of a PRP clot, whereas endothelialcells are plated within the PRP clot in a seeded graft.

The underlying hypothesis is fairly simple; that is, by promoting theestablishment of the patient's own endothelial cells on the bloodcontacting surface of a vascular prosthesis, a “normal” endothelial celllining and associated basement membrane, together known as theneointima, will form on the graft and counteract the rheologic,physiologic, and biomaterial forces working synergistically to promotegraft failure. After 30 years of research in this area, includingpromising animal data, this simple hypothesis has not yet yielded aclinical device.

The failure modes with endothelial-seeded grafts have been identical tountreated polymeric grafts, namely thrombosis and intimal hyperplasia.The failure modes, at least partially, are linked to the lack of afunctional endothelial layer, neointima, on the luminal surface of thegraft and/or abnormal endothelial and smooth muscle cell direct andindirect communication. These failures in early human trials camedespite successful demonstrations of seeded grafts developing into acell lining development. These data show that neo-intimal formation onpolymeric vascular graft lumenal surfaces in animal models occurs byendothelial cell proliferation from perianastomotic arteries, themicrovessels of graft interstices, or circulating progenitor endothelialcells not strictly from the seeded cells.

A potential source for endothelial cell seeding is microvascularendothelial cells (MVEC). Williams et al. pioneered both freshlyisolated and cultured human, canine, rabbit, rat, bovine and pigendothelial cells, specifically MVEC, in their laboratory to studycellular function. The source for human MVEC was aspirated tissue fromcosmetic liposuction. Two separate protocols for human fat MVECisolation were used depending on the end use of the cell population. Theprotocols differed in isolation complexity from a simple, operatingroom-compatible procedure for immediate sodding of human or animalgrafts to a more elaborate procedure if the MVEC will be subsequentlycultured.

A human clinical trial was undertaken to evaluate endothelial celltransplantation in patients requiring peripheral bypass. During thetrial, large quantities of endothelial cells were placed directly on thelumenal surface of an ePTFE graft. To improve cell deposition, allgrafts were pre-wetted in culture medium containing autologous serum.Cells were suspended in the same medium at a density of 2×10⁵ cells/cm²graft lumenal area. This solution was held at a cross-wall, ortransmural, pressure gradient of 5 psi to force cells onto the surface,a process termed “pressure sodding”. After institutional approval, 11patients were enrolled and received the experimental graft. Duringsurgical prep, the patients underwent liposuction to removeapproximately 50 grams of abdominal wall fat. The fat was processedusing the aforementioned procedure and the resulting cell population waspressure sodded on the intended graft and immediately implanted. Aftermore than 4 years of follow-up, these grafts have maintained a patencyrate similar to that of saphenous vein grafts.

Pressure gradients involving transient (<1 min.) relatively highpressures (250 mmHg) have previously been used to deposit cells onto apermeable scaffold by a sieving action, i.e., providing a bulk flow andusing a substrate or scaffold material having pores smaller than thecell population, thus capturing cells in the matrix. However, despitethe aforementioned advances, clinical coronary applicability has beenlimited to date because the vessels do not maintain sufficientlycohesive non-thrombogenic surfaces; research has focused on additionalmaturation time in vitro.

Endothelial cells are of critical importance in establishing anon-thrombogenic cell lining. In addition, a need still exists for anefficient and reliable method for producing endothelial cell linings ona synthetic graft in an operating room setting, and the currentdisclosure provides a solution. It is desirable to achieve rapid celladhesion in or on a permeable matrix, scaffold or other permeable cellsubstrate material in a matter of minutes or hours with an instrumentthat lends itself to the operating room environment, maintains a sterilebarrier, is easy to use, produces consistent graft results, and isinexpensive.

LISTING OF THE FIGURES

FIG. 1 is a partial isometric view of a cell separation system 100according to certain embodiments of the present disclosure.

FIG. 2 is a partial isometric view of a single-walled bowl assembly 102according to certain embodiments of the present disclosure.

FIG. 3 is a partial exploded view of a single-walled bowl assembly 102according to certain embodiments of the present disclosure.

FIG. 4A is a front view of a seamed single-walled bowl according tocertain embodiments of the present disclosure.

FIG. 4B is a partial side view of a single-walled bowl according tocertain embodiments of the present disclosure.

FIG. 4C is a partial top view of a single-walled bowl according tocertain embodiments of the present disclosure.

FIG. 4D is a partial cross-sectional view of a single-walled bowl takenacross the bowl through the pellet concentration areas according tocertain embodiments of the present disclosure.

FIG. 4E is a partial cross-sectional view of a single-walled bowl takenacross the bowl perpendicular through the pellet concentration areasaccording to certain embodiments of the present disclosure.

FIG. 5A is a top view of a cradle with the lid closed according tocertain embodiments of the present disclosure.

FIG. 5B is a partial side view of a cradle with the lid closed accordingto certain embodiments of the present disclosure.

FIG. 5C is a partial front cross-section view of a cradle with the lidclosed according to certain embodiments of the present disclosure.

FIG. 5D is a partial isometric view of a cradle with the lid openaccording to certain embodiments of the present disclosure.

FIG. 5E is a partial isometric view of a cradle with the lid closedaccording to certain embodiments of the present disclosure.

FIG. 6 is a method of separating endothelial cells from biologicmaterial such as adipose tissue according to certain embodiments of thepresent disclosure.

SUMMARY

An embodiment of the present disclosure describes a method of separatingcells, comprising agitating, in a reservoir of a cell separation system,a volume of biologic material and a volume digestion media to form adigested volume of biologic material; centrifuging the digested volumeof biologic material at a force from about 500 G to about 1000 G fromabout 5 minutes to about 10 minutes to separate the digested volume intoa plurality of concentrated cell volumes and a plurality of waste;removing a portion of the plurality of waste via at least one fluidoutlet; isolating a different portion of the plurality of waste, whereinthe different portion of the plurality of waste comprises waste with alarger diameter than the at least one fluid outlet and is not in contactwith the concentrated cell volumes; and removing the concentrated cellvolumes from the reservoir.

An embodiment of the present disclosure describes a cell separationsystem, comprising a non-transitory storage device comprising aplurality of logic associated with centrifugation programs, whereinexecution of a centrifugation program separates a cell volume from abiologic material volume; a heating mechanism electrically coupled to apower supply; a containment mechanism in proximity of the heatingmechanism; an assembly removably coupled to the containment mechanism,wherein the assembly comprises a single-walled centrifugation bowlcomprising a plurality of cell concentration areas, a reservoir, and acenter column comprising a plurality of fluid lines, a cradle, whereinthe cradle comprises a plurality of apertures formed concentricallythrough the cradle, wherein the bowl is removably coupled to the cradleto enable free rotation of the bowl while coupled to the cradle, analignment mechanism coupled to the containment mechanism and the bowl torestrict movement of the center column when the first assembly iscoupled to the containment mechanism, wherein, in a first state of acentrifugation program, the assembly is configured to rotate around acentral axis in a first direction and in a second direction in analternating fashion and the heating mechanism is activated, and wherein,in a second state of a centrifugation program, the heating mechanism isdeactivated and the single-walled bowl is configured to rotate in asingle direction around the central axis to separate a plurality ofwaste from a plurality of cells.

An embodiment of the present disclosure describes a cell separationsystem comprising a non-transitory storage device comprising a pluralityof logic associated with a plurality of different centrifugationprograms, wherein, when executed by a processor; agitates, via aplurality of paddles of the system, a volume of biologic material and avolume digestion media to form a digested volume of biologic material,wherein the volume of biologic material and the volume digestion mediaare agitated via the plurality of paddles in a reservoir of the system;separates, via centrifugation, the digested volume of biologic materialat a force from about 500 G to about 1000 G from about 5 minutes toabout 10 minutes to separate the digested volume into a plurality ofconcentrated cell volumes and a plurality of waste; removes, based onthe program, subsequent to centrifugation a portion of the plurality ofwaste via at least one fluid outlet; isolates a different portion of theplurality of waste, wherein the different portion of the plurality ofwaste comprises waste with a larger diameter than the at least one fluidoutlet and is not in contact with the concentrated cell volumes; andremoves the concentrated cell volumes from the reservoir.

DETAILED DESCRIPTION

Endothelial cells are used to establish non-thrombogenic cell liningwithin synthetic grafts. Thus, it is desirable to achieve rapid cellularadhesion in or on a permeable matrix, scaffold, or other permeable cellsubstrate material in a matter of minutes or hours with an instrumentthat lends itself to the operating room environment, maintains a sterilebarrier, is easy to use, and produces consistent graft results.

Currently, there are various approaches for meeting these requirements,but with limited success: (i) the use of decellularized tissuematerials; (ii) the use of a self-assembly mechanism, wherein cells arecultured on tissue culture plastic in a medium that inducesextracellular matrix (ECM) synthesis; (iii) the use of syntheticbiodegradable polymers, onto which cells are subsequently seeded andcultured in a simulated physiological environment; and (iv) the use ofbiopolymers, such as a reconstituted type I collagen gel, which isformed and compacted with tissue cells by the application of mechanicalforces to simulate a physiological environment.

Embodiments of this disclosure describe systems and methods that enablethe isolation of large quantities of endothelial cells from fat tissueand the rapid cell sodding of synthetic grafts, and that enable theautomation and adhesion of cells in a turn-key, operating room readyinstrument for the rapid sodding of the graft. Embodiments of thisdisclosure likely have other applications in addition to the lining ofgrafts for implantation.

The systems and method discussed herein are of a cell separation systemthat comprises a single-walled bowl that may be formed as a single piece(no seams or welds) or which may be formed as a plurality of pieces. Thesingle-walled bowl (“bowl”) may comprise various areas including acenter column that houses a plurality of tubing that is used as fluidinlets, waste outlets, and extraction of the separated cells via a cleanline that has not been contaminated by waste. The bowl further comprisesa plurality of cell concentration areas disposed circumferentiallyaround a central axis of the bowl such that, during centrifugation, thegravitational forces separate the cells from a biological materialvolume and store the cells until the waste has been collected as to notcontaminate the separated cells. The bowl couples to a cradle and iscoupled via a plurality of locking features and mechanisms that may bemechanical, magnetic, electrical, or a combination of mechanisms. Analignment mechanism is coupled to the bowl such that it engages with thecontainment mechanism in which the assembly of the bowl and the cradleis disposed.

In one example of cell separation using the cell separation systemdiscussed herein, a plurality of logic is stored in a non-transitorystorage device (memory) and comprises a plurality of centrifugationprograms. Each program of the plurality of programs comprisesinstructions that may be based upon a plurality of factors includingmedia type and biological material volume and/or target cell volume ortarget cell concentration. When executed by a processor, each programinitiates cell separation through a plurality of states as discussedherein, resulting in the automated removal of the separated cells andthe trapping and/or removal of waste. The programs may differ and/oroverlap in various aspects, including temperatures, times, forces, andoverall program length (time) from disposal of the biologic materialvolume and the media until the removal of the separated cell volume.

In one example of the cell separation system, a plurality of digestionmedia, and a plurality of biologic material (a volume) are disposed inthe bowl, in particular in a reservoir of the bowl. The bowl is thenagitated, rotated in each direction around the central axis to break upthe biologic material volume to enable the separation duringcentrifugation. The agitation may be promoted by a plurality of positivefeatures that may be referred to as paddles or fins. In one example,these features are formed integrally with the bowl, and in anotherexample they may be removable, separate components. The bowl may beheated prior to and/or during the agitation via a heating mechanism, theheat generated by the heating mechanism causes the air in a gap betweenthe cradle and the containment mechanism to heat up, and the pluralityof apertures in the base of the cradle enable the circulation of thisheated air. The bowl may be heated from about 25 C to about 45 C, andthe heating mechanism may be shut off subsequent to completion ofagitation such that the remainder of the cell separation occurs at roomtemperature (from about 20° C. to about 25° C.).

Subsequent to the agitation, centrifugation is performed. Duringcentrifugation, the cell separation assembly of the bowl and cradlerotates freely with respect to the containment mechanism and analignment mechanism. The centrifugation may be performed at a force fromabout 500 G to about 1000 G for from 5 minutes to 20 minutes, or from 1minute to 10 minutes, or other ranges of time in various embodiments.Cells are separated from the mixture in the reservoir and aregravitationally forced into a plurality of cell concentration areas ofthe bowl. After the centrifugation, the speed of the bowl is reduced to10%-30% of the average rotation force during centrifugation, and aplurality of waste materials are removed via a plurality of fluid lines.Waste that has a diameter larger than the fluid lines (fluid outlets) istrapped in a waste region and does not contact the separated cellvolume. The rotation may be stopped, causing the collected cells in thecell concentration areas to slide down the sides of the bowl and intothe reservoir where the cells are removed via the clean line. In otherexamples, a volume of media less than 10 mL or 5 mL is jet-sprayed intothe cell concentration areas in order to remove any remaining cells. Byusing a single-walled bowl, the heat transfer may be more effective,thus increasing the efficiency of the system and reduces the costassociated with cell separation.

FIG. 1 is a partial isometric view of a cell separation system 100according to certain embodiments of the present disclosure. The cellseparation system 100 comprises an assembly 102 of a single-walledcentrifugation bowl 104 coupled to a cradle 108 via a plurality ofcoupling mechanisms including a first 104A and a second 104B lidportion. The assembly 102 is configured such that an alignment mechanism122 acts to stabilize the assembly 102 and allow free rotation of theassembly 102 (the bowl 302 as coupled to the cradle 108). Duringcentrifugation, the assembly 102 spins in either direction 130A or 130Baround a central axis 124, shown in a coordinate system in FIGS. 1 and 3and referenced throughout. The coordinate system further comprises asecond axis 126 that is perpendicular to the central axis 124, and athird axis 128 perpendicular to both 124 and 128. The cradle 108 isseated in and removably coupled to a containment mechanism 110, whichacts to direct the heat and hot air generated by the activation of theheating unit 116. The coupling of the containment mechanism 110 to theassembly 102 creates an air gap between the two through which hot airmay be circulated via an open bottom of the cradle 108 (not shown) andthrough the plurality of apertures 106 in the cradle 108. A hot airblower 112 also operates to circulate air in the system. In anembodiment the single walled bowl assembly 102 and associated tubing aredisposable.

In an embodiment, the heating unit 116 is employed to elevate atemperature of the system 100 during at least agitation. The heatingunit 116 is coupled to a base 114 comprising a plurality of feet 120configured to prohibit movement of the base 114 and system 100 duringexecution of a plurality of centrifugation programs. The heating unit116 may be wired or wireless, and, if wired via 118, may be coupled to apower source during use and/or to charge a wireless battery or batteriescontained in the heating unit 116. The heating unit 116 furthercomprises a plurality of heating elements configured to elevate atemperature of the containment mechanism 110 and thus the assembly 102coupled to the mechanism 110. The containment mechanism 110 may also becoupled or removably coupled to the base 114 as well.

In an embodiment, the system 100 may comprise at least one storagedevice (not shown) comprising a plurality of centrifugation programs anda processor, as well as a plurality of controls (not shown) activated bythe execution of a centrifugation program via the processor. The storagedevice and/or the plurality of controls that may be located on thesystem 100 or located remotely and accessed via a tablet, mobile phone,wearable technology, kiosk, laptop computer, or desktop computer. Eachcentrifugation program may comprise a plurality of parameters employedin the centrifugation cycle, and may be selected manually or dynamicallyand in an automated fashion based upon inputs such as the biologicmaterial volume used and/or the volume and/or type of digestionenzyme(s) employed.

FIG. 2 is a partial isometric view of a single-walled bowl assembly 102according to certain embodiments of the present disclosure. FIG. 2illustrates the bowl-to-cradle locking mechanism 202 that includes thefirst 104A and second 104B lids as well as components discussed below.The feature 202 is referred to herein as a locking mechanism 202 togenerally refer to the coupling of the lid portions 104A and 104B to thecradle 108 via the “edge” features of the locking mechanism 202 thatalign and are removably coupled as discussed in detail in FIG. 3. Inanother embodiment, the locking mechanism 202 is comprised of a magneticlatch or magnetic catching mechanism removably coupled to the bowlassembly 102.

FIG. 3 is a partial exploded view 300 of a single-walled bowl assembly102 according to certain embodiments of the present disclosure. FIG. 3shows the first lid portion 104A and the second lid portion 104B, aswell as the associated first portions 202A of the locking mechanism 202that are removably coupled to the second portions 202B that are a partof the cradle 108. The single-walled bowl 302 is shown here, this bowl302 may be fabricated as a single, seamless piece or as multiple piecesassembled into the bowl 302, as discussed below. The “single-wall” ofthe bowl is in contrast to a bowl that has at least two nested walls.

In an embodiment, the bowl 302 comprises a first shoulder 304A thatcouples to the first lid portion 104A and a second mating lid portion312A, and a second shoulder 304B along a shared axis that isperpendicular to a central axis 124 and that couples to the second lidportion 104B and a second mating lid portion 312B. The lid portions104A, 104B, the mating lid portions 312A, 312B, and the first 202A andsecond 202B portions of the locking mechanism 202 (formed when 202A and202B are removably coupled via mechanical, magnetic, or other means) actto secure the bowl 302 in the cradle 108 during operation. Once secured,the assembly 102 rotates freely with respect to the containmentmechanism, which does not rotate and acts at least in part to direct andcirculate heated air towards the bowl 302 during agitation (andbreakdown) of the biologic material volume.

In an embodiment, the bowl 302 further comprises a first 306A and asecond 306B cell concentration area that each comprise smooth internalgeometries and act to isolate and concentrate, via the removal of fluidand solids, the cells separated during centrifugation. A cradle matingportion 308 of the bowl 302 may extend downward into and be seated inthe cradle, as discussed below. A center portion of the bowl 302including a collar 312 houses the alignment mechanism 122 and aplurality of tubing and access points (not shown here). The alignmentmechanism 122 prevents the bowl 302 and cradle 108 from spinning orrotating while interfacing with the containment mechanism 110.

In an embodiment, the bowl 302 further comprises an indentation 310 or aplurality of indentations 310 shown on the outside of the bowl 302 inFIG. 3 but formed using the wall of the bowl 302 such that an internalrib or ribs are formed by the indentation and on the interior surface ofthe bowl 302. This rib or ribs extend into a chamber area (not shown inFIG. 3) where the digestion occurs, and thus may be referred to as adigestion chamber of the bowl 302, and the rib or ribs act to promotedigestion by agitation of the bowl 302 as discussed in the method inFIG. 6. The positive features formed by the indentations 310 on theinterior of the bowl 302 may be referred to as paddles or ribs and arediscussed further below.

FIG. 4A is a first view 400A of a seamed single-walled bowl 302according to certain embodiments of the present disclosure that may bereferred to as a partial front view 400A. The view 400A of the bowl 302is taken parallel to the axis 128 in FIG. 3. As shown in FIG. 4A, thebowl 302 may comprise an at least one seam 422 along a centerline of thebowl 302. Also shown in FIG. 4A are a plurality of attachment mechanisms402 that removably couple to the alignment mechanism (122 notillustrated here) and a center column or cavity 404 that retains aplurality of tubing discussed below configured to introduce thebiological material volume and clean fluids and remove waste fluids andsolids.

FIG. 4A additionally illustrates the first and second shoulders 304A and304B and the cell concentration areas 306A and 306B, all of which areformed as integral parts of the bowl 302 in an example where there is noseam 422, such as that shown in FIG. 3. The alignment mechanism 122(FIG. 3) is removably coupled to the containment mechanism 110 andprevents the center column 404 from moving, thereby permitting theassembly 102 to spin relative to the containment mechanism.

FIG. 4B is a partial side view 400B of a single-walled bowl 302according to certain embodiments of the present disclosure. FIG. 4B is aview perpendicular to the axis 128 as shown in the inset in FIG. 3. FIG.4B shows features illustrated in FIGS. 1-4A, including the collar 312,first concentration area 306A, the plurality of attachment mechanisms402, the center column 404, the shoulder 304A, and a rounded bottomportion 424. The rounded bottom portion 424 of the bowl 302 is seated inthe cradle (not shown here) and is a smooth, continuous surface.

FIG. 4C is a partial top view 400C of a single-walled bowl according tocertain embodiments of the present disclosure. FIG. 4C is a view takenperpendicular to the axis 124 as shown in the inset in FIG. 3. FIG. 4Cillustrates a plurality of fluid lines 406, 408, 416 and a waste outlet418, discussed in detail below. A plurality of tubing and/or syringe orother collection and introduction vessels may be coupled to the entryports of the lines 406, 408, 416, and 418, depending upon the functionsof each line. In various embodiments, some fluid lines are employed tointroduce biological material, some introduce media such as digestionenzymes, some may introduce other media, at least one is employed forwaste removal, and at least one is a clean line used for the removal ofthe separated cells so that the cells are not contaminated by the wastefluid or tissue.

FIG. 4D is a partial cross-sectional view 400D of a single-walled bowltaken across the bowl through the pellet concentration areas accordingto certain embodiments of the present disclosure. FIG. 4D is a viewtaken perpendicular to the 128 axis as shown in the inset in FIG. 3.FIG. 4D illustrates the bowl chamber 428, which is the interior of thebowl 302. The bowl chamber 428 comprises at least the concentrationareas 306A and 306B, the reservoir 426, a waste block 420, center column404, and a plurality of agitation paddles 414. A plurality of fluidtransport tubes and ports are contained in the center column 404. Thesemay include a jet spray inlet 406 configured as a media inlet, first 408and second 416 fluid lines, each configured to introduce media or otherfluids or biologic materials and to withdraw media or biologicmaterials. If one of the first 408 and second 416 lines are employedsolely to introduce media, this line may be referred to as the “clean”line. That is, once either of lines 408 or 416 are used to withdrawfluids or solids from the bowl 302, that line may not be used tointroduce clean media or other fluids or biologic materials to preventcontamination. Each of the lines 406, 416, and 408 (FIGS. 4C and 4E) maybe coupled to flexible tubing extending from a top surface of the bowl302.

In some examples, a syringe or other sterile collection device may becoupled to one of 408 and 416, depending upon which is designated as theclean line. This sterile collection device is removably coupled towhichever line is the clean line since that line is used to remove theconcentrated cells after separation. Also shown in the center column 404is a waste outlet 418 also called a skimmer. The waste outlet 418 mayhave a larger diameter than the fluid lines 408 and 416 and may be ableto remove larger solids. Also shown in FIG. 4D are the paddles 414 whichmay be formed from the indentations 310 from FIG. 3 or which may beformed separately. Two or more paddles 414 may be employed for agitationas discussed in FIG. 3. Also shown in FIG. 4D are access points 408A and416A.

The waste block 420 is configured to isolate (trap) solids that are toolarge to be removed via the lines 416 and 408. By trapping solids thatare too large to exit via a fluid line, these particles are prohibitedfrom clogging the system. The smooth interior surface 412 of the bowl302 is also shown, it is along here where the cells collected in theconcentration areas 306A and 306B are released when centrifugation isstopped or slowed, the cells are retained in the reservoir 426 andremoved through the clean line, which is one of 408, 416, or 406. Thewaste block 420 is fluidly connected to the fluid lines 408 and 416 andmay be described as an inverted conical shape.

FIG. 4E is a partial cross-sectional view 400E of a single-walled bowltaken across the bowl perpendicular through the pellet concentrationareas according to certain embodiments of the present disclosure. FIG.4E is a view taken perpendicular to the 128 axis as shown in the insetin FIGS. 4A and 4B. FIG. 4E illustrates the fluid lines 416 and 408 thatextend to the bottom of bowl chamber 428 but do not contact the interiorsurface of the bottom of the bowl chamber 428, such that the lines 416and 408 introduce and/or remove materials to and/or from the reservoir426.

FIG. 5A is a partial front view 500A of a cradle 108 with the lid closedaccording to certain embodiments of the present disclosure. FIG. 5Aillustrates a cradle base 514 comprising the plurality of apertures 106,these apertures 106 act to release heat generated during cell separationcycles. A cradle neck 508 extends from the base 514 to the first 104Aand second 104B lid portions and the locking mechanism 202. Asingle-walled bowl as described herein is secured to the cradle 108 viathe lid portions 104A and 104B and the locking mechanism 202, and itrests in part in the recess 502 formed in the neck 508 and defined by afirst dimension 512 and a second dimension 510, as well as a depth (notshown here).

FIG. 5B is a partial side view 500B of a cradle 108 with the lid closedaccording to certain embodiments of the present disclosure. FIG. 5C is apartial front cross-section view 500C of a cradle 108 with the lidclosed according to certain embodiments of the present disclosure. FIGS.5B and 5C are discussed interchangeably herein. FIG. 5B illustrateselements previously discussed as well as a cradle edge 602 which extendsaround the outside diameter of the base 514 and connects to the neckregion 508. When the lid is closed, as is pictured in FIG. 5B, the lidportions 104A and 104B are coupled to the cradle 108 via the firstportions 202A that extend from each lid 104A and 104B, respectively, andare removably coupled to the second portions 202B to form the lockingmechanism 202 via mechanical, magnetic, or other means. FIG. 5Badditionally shows aperture 516 that extends through the cradle 108 andis where the shoulders (304A and 304B from FIG. 3) are secured when thebowl is coupled to the cradle 108.

FIG. 5D is a partial isometric view 500D of a cradle with the lid openaccording to certain embodiments of the present disclosure. In FIG. 5D,the locking mechanism portions 202A of the lids 104A are partiallycoupled to the locking mechanism portion 202B of the cradle 108 at hingeregions 518. The aperture 516 shown in FIG. 5B is formed during lidclosure by 312A, 312B, and of each of the first 104A and second 104B lidportions when the first locking mechanism portion 202A is partiallycoupled to the second locking mechanism portion 202B. As used herein, a“partial coupling” comprises a hinged coupling 518 as shown in FIG. 5D,and a “full coupling” comprises a closed lid as shown in at least FIGS.5A-5C and FIG. 5E.

FIG. 5E is a partial isometric view 500E of a cradle 108 with the lidclosed according to certain embodiments of the present disclosure. FIG.5E illustrates the lid closed such that the locking mechanism 202 isengaged, which secures the bowl (not shown) in place and the aperture516 is formed.

FIG. 6 is a method of separating endothelial cells from biologicmaterial according to certain embodiments of the present disclosure. Themethod 600 is an automated mechanism for the washing, separation,concentration, and removal/harvesting of viable cells such asendothelial cells. The harvested cells may be employed for grafts orotherwise employed for use in healthcare (e.g., FDA approvedimplantation or further processing) and/or healthcare research (trialsto approve cell separation methods and cell-derivative products foruse).

In an embodiment, at block 602 of the method 600, a biologic materialvolume is disposed in a reservoir 426 (FIG. 4D) of a single-walled bowlof a cell separation system such as the system 100 in FIG. 1. At block604, a plurality of media and a collagenase enzyme or enzymes may beintroduced into the chamber 302 in particular into the reservoir 426 viaany or all of the lines 406, 408, or 416 as discussed above with respectto clean lines. The media may be fed into the system at block 604.Examples of media may be Lactated Ringers (LR), Hartmans, Water ForInjection (WFI) or any other similar media. In some examples, thereservoir 426 may be pre-heated to from about 30° C. to about 40° C.prior to disposal of the biologic material volume and/or media in thechamber. In some examples, block 602 may occur prior to block 604, andin other examples, block 604 may occur prior to block 602. In stillother examples, blocks 602 and 604 may occur near-simultaneously suchthat the biologic material volume and media are disposed in thereservoir 426 at approximately the same time. In an example where thereservoir 426 is heated, it may be heated to and maintained at atemperature from about 30° C. to about 40° C. prior to block 602, block604, or after either or both of the biologic material volume and/ormedia are disposed in the reservoir 426. This heating may be referred toas a pre-heating, which may take from about 10 minutes to about 45minutes, or less than 10 minutes depending upon a target temperature ortemperature range.

Further in the method 600 at block 606, the biologic material volumedisposed at block 602 is completely or partially digested via the enzymemedia disposed at block 604. The digestion at block 606 of the biologicmaterial volume disposed at block 602 may occur via heating of thereservoir 426, or via the agitation of the single-walled bowl, or by acombination of both. This digestion may occur over various time periodsfrom 5 minutes to 1 hour, from 15 minutes to 45 minutes, or otherperiods of time depending upon a type of enzyme(s) used, a number ofenzyme(s) used, and/or a volume of each enzyme used at block 604, andthe volume of biologic material disposed at block 602. As discussedherein, the agitation of the single-walled bowl causes the paddles 414to engage with the media and biologic sample and create agitation thatpromotes and enables digestion. The agitation at block 606 may occur bythe partial or complete rotation of the bowl in alternating directionsaround a central axis, and is not the same as, nor does it generate theforce of, the centrifugation discussed herein. The agitation employed topromote digestion at block 606 may comprise whole or partial rotationsin different directions around a central axis 124 (FIG. 1), and mayoccur for a predetermined period of time. The digestion at block 606 maybe referred to as a first state of the apparatus, wherein centrifugalforces are not applied and the media and biologic material volume areagitated in the chamber for a predetermined period of time or for apredetermined number of agitation cycles. In one example, thetemperature of the reservoir 426 is maintained at about 37° C. duringthe digestion at block 606, this first state is maintained for apredetermined time period based upon a type of enzyme(s) used, a numberof enzyme(s) used, and/or a volume of each enzyme used at block 604, andthe volume of biologic material disposed at block 602. The systemsdiscussed herein may comprise a plurality of stored programs comprisingparameters for the digestion at block 606 as well as for other states,blocks, and phases discussed herein.

At block 608, in response to and subsequent to the completion ofdigestion at block 606, centrifugation occurs. As discussed herein, the“completion” of the digestion at block 606 refers to when the digestionhas progressed to a point where the cells are still viable but thebiologic volume has been broken down such that it is capable ofcentrifugation at block 608. This centrifugation at block 608 may becharacterized by the separation of endothelial cells from the digestedvolume formed at block 606. In an embodiment, the configuration at block608 comprises a g-force from 600 G to 1000 G for a time period fromabout 5 minutes to about 10 minutes. The g-forces or “G” referred toherein is the force of gravity applied to a body, in this case, theforce applied to the cells collected in the cell concentration areaswhich continue to have fluid removed (thus becoming a more concentratedcell volume with the reduced fluid/waste) and remain isolated from thereservoir during at least the centrifugation at block 608.

The centrifugation at block 608 may be referred to as a second state ofthe cell separation system. The centrifugation at block 608 may compriseprograms of various RPM speeds and times for cycles. These cycles mayincrease in RPM and/or in duration until cessation at block 612 asdiscussed below. In an embodiment, a single centrifugation cycle may beemployed from 500 G to 1000 G for a time period from about 5 minutes toabout 10 minutes and in still other examples, multiple centrifugationcycles may be employed that first increase in the G-force applied andthen decrease the force applied, leading into the slowed rotationdiscussed in detail in block 610 below.

In an embodiment, the digestion at block 606 separates a plurality ofcells from adipose tissue and fluids, the centrifugation cycle(s) atblock 608 acts to force the separation of the cells and the movement ofthose separated cells into the cell concentration areas 306A and 306B asshown in FIG. 3. In an embodiment, the heating mechanism used to preheatthe reservoir 426 during digestion at block 606 is shut off subsequentto block 606 and prior to the initiation of block 608, such that thecentrifugation at block 608 may proceed between room temperature (fromabout 20° C. to about 25° C.) and the temperature employed at block 606.Further at block 608, during separation of the cells into the cellconcentration areas 306A and 306B, after a predetermined time period ofcentrifugation at block 608, clean media may be introduced into thechamber of the single walled bowl via the line 408. This media furtherdisplaces a plurality of materials including fat and other tissues andliquids from the reservoir 426 such that those materials are removedfrom the reservoir 426 as the volume increases via the line 418 that maybe coupled to a waste container or other vessel. In some examples, aspeed of from 500 G to 800 G may be used at block 608 in order toseparate cell volume in the concentration areas 306A and 306B, and inother examples, a speed from 700 G to 900 G may be used forcentrifugation at block 608. In some embodiments, at block 608, duringcentrifugation, a plurality of clean media may be introduced via a fluidline such as 408. The clean media may be added at block 608 to increasethe total volume inside the bowl 302 to promote the expulsion of waste,which floats in the media.

Block 610 is a slowed rotation phase and may also be referred to as thethird state of the cell separation apparatus. During this phase, whichmay be from 3 minutes to 45 minutes, a lower centrifugal force, forexample, from about 15 G to about 50 G, and in some cases from 20 G to25 G, is applied at block 610. In one example, at block 610, therotation of the single-walled bowl is slowed to a predetermined speed orrange of speeds, and a temperature of the chamber may be from roomtemperature to the maximum temperature employed at block 606. Theparameters such as speed (force generated) and temperature may beemployed at block 610 to enable the separated cell volume to remain inthe cell concentration areas 306A and 306B while the remaining fluidfrom the media and biological volume drains down the interior walls ofthe chamber.

The waste collected may be removed in whole or in part via the wasteoutlet 418 or fluid line 416 while the cell volumes are retained in thecell concentration areas 306A and 306B by the centrifugal force appliedat block 610. In some examples, a minimum force applied at block 610 maybe about 20 G, and a maximum force may be about 50 G. Some waste mayremain trapped in 420, where it is isolated from the separated cells. Insome examples, the slowed spin of block 610 may be iterative, forexample, a first slowed rotation at block 610 may be at 90% of anaverage speed of block 608, a second, subsequent slowed rotation atblock 610 may be at 80% of an average speed of block 608, and subsequentslowed rotations may be at lesser and lesser speeds until apredetermined period has expired or until a predetermined amount offluid and/or solids has been removed, as determined by volume and/oroptic sensors. During block 610, fluid and solids that are small enoughto fit through the waste outlet 418 or fluid line 416 are removed andany solids larger than those tubes are retained and isolated by thewaste block 420 such that the separated cell volume is not contacted bythis material.

At block 612, the rotation is slowed to a stop. The slowing andcessation at block 612 of the rotation causes the cells collected to nolonger be compacted in these regions since the force holding the cellsin place and concentrating the cell volume (removing liquid) is removed.The normal gravitational force may then be employed to collect cellsfrom the collection areas 306A and 306B to a collection region of thesingle-walled bowl. Thus, the cells collected in 306A and 306B move fromthose collection areas to a receptacle. At block 614, the concentratedcells (separated cell volume) may be removed via the clean line 408 viaa syringe or other mechanism, this removal is automated and occurs inresponse to completion of blocks 610 and 612. Blocks 612 and 614 may becollectively referred to as a fourth state of the cell separationapparatus when the bowl is no longer rotating relative to the cradle.

In some examples, at block 616, prior to or simultaneously with thecessation of rotation and removal of cells at blocks 612 and 614,respectively, a wash media is introduced to the chamber. In thisexample, the introduction of media at block 616 may occur depending uponthe rotation speeds, centrifugation programs (cycles), and geometry ofthe system and collection regions. In an embodiment where wash media isintroduced at block 616, the amount employed may be less than 10 mL, 5mL, or 1 mL, and the wash media is used to ensure that the cellscollected in the concentration regions are dislodged and removed atblock 616 so that they can be collected at block 614. In this example,block 616 may also be considered as a part of the fourth state of thecell separation apparatus. The blocks discussed in the method 600 areassociated with an automated, dynamic method of cell separation andcollection, such that the loading of the chamber at blocks 602 and 604proceeds through the removal and collection at block 612 without manualintervention. In one example, a non-transitory memory stored on astorage device and coupled to the cell separation apparatus comprises aplurality of code executable by a processor. This plurality of codecomprises centrifugation programs for samples of varying properties,each program may comprise a flow rate for blocks 602 and/or 604, as wellas cell volume and/or concentration targets, flow rates, times, andranges for forces generated (rotation rate/RPM) at blocks 606, 608, 610,614, and 616 as appropriate for the actions occurring at each block.Each program may be associated with an overall time to completion fromthe deposition of the media and biologic material volume to the removalof the separated cells.

While preferred embodiments have been shown and described, modificationsthereof can be made by one skilled in the art without departing from thescope or teachings herein. The embodiments described herein areexemplary only and are not limiting. Many variations and modificationsof the systems, apparatus, and processes described herein are possibleand are within the scope of the invention. For example, the relativedimensions of various parts, the materials from which the various partsare made, and other parameters can be varied. Accordingly, the scope ofprotection is not limited to the embodiments described herein, but isonly limited by the claims that follow, the scope of which shall includeall equivalents of the subject matter of the claims. Unless expresslystated otherwise, the steps in a method claim may be performed in anyorder. The recitation of identifiers such as (a), (b), (c) or (1), (2),(3) before steps in a method claim are not intended to and do notspecify a particular order to the steps, but rather are used to simplifysubsequent reference to such steps.

The invention claimed is:
 1. A cell separation system, comprising: a non-transitory storage device comprising a plurality of logic associated with centrifugation programs, wherein execution of a centrifugation program separates a cell volume from a biologic material volume; a heating mechanism electrically coupled to a power supply; a containment mechanism in proximity of the heating mechanism; an assembly removably coupled to the containment mechanism, wherein the assembly comprises: a single-walled centrifugation bowl comprising a plurality of cell concentration areas, a reservoir, and a center column comprising a plurality of fluid lines, a cradle, wherein the cradle comprises a plurality of apertures formed concentrically through the cradle, wherein the bowl is removably coupled to the cradle to enable free rotation of the bowl while coupled to the cradle, an alignment mechanism coupled to the containment mechanism and the bowl to restrict movement of the center column of the bowl when the first assembly is coupled to the containment mechanism, wherein, in a first state of the centrifugation programs, the assembly is configured to rotate around a central axis in a first direction and in a second direction in an alternating fashion and the heating mechanism is activated, and wherein, in a second state of the centrifugation programs, the heating mechanism is deactivated and the single-walled bowl is configured to rotate in a single direction around the central axis to separate a plurality of waste from a plurality of cells.
 2. The system of claim 1, wherein the separated cells are separated in the second state of a centrifugation program into a plurality of cell concentration areas in the single-walled bowl.
 3. The system of claim 1, further comprising a third state, wherein, when the system is configured in the third state, the system is configured to drain a portion of the plurality of waste via a waste outlet.
 4. The system of claim 3, wherein, when configured in the third state, the system traps, in a waste block formed in the single-walled bowl, another portion of the plurality of waste comprising an average diameter larger than the waste outlet.
 5. The system of claim 1, wherein, when configured in a fourth state, the single-walled bowl is stationary.
 6. The system of claim 1, wherein the plurality of fluid lines comprises a waste outlet and a clean line. 